The ability to detect and quantify nucleic acid polymorphisms is useful in the characterising of individual animals, humans, plants and other organisms. In particular, it is desirable to detect a difference in a nucleic acid polymorphism to distinguish between individuals.
Methods for determining the presence of nucleic acid target sequences, in particular a base at a target position in a sample of DNA are known. One such method is to separate DNA fragments of interest in a sample by methods such as agarose or acrylamide gel electrophoresis and hybridise a labeled oligonucleotide probe to a nucleic acid sequence in a sample or on a blot (Southern blotting) and to then detect the presence of the hybridised probe via the label. Generally the label is a radioactive material, fluorescent dye or enzyme such as horseradish peroxidase, urease or alkaline phosphatase.
Another method for the detection of the hydridised probe and nucleic acid sequence is the use of polymerase chain reaction (PCR) techniques. This method requires the use of two (2) oligonucleotides, the PCR primers. These PCR primers are annealed to the target sequence in a cyclic manner and the DNA polymerase reaction extends the DNA duplex from the primers resulting in the amplification of the region between the primers. The absence of a PCR product indicates that hybridisation of one or both primers did not occur. Detection of the PCR product may be achieved in similar ways as described above, although methods are available that rely on non-gel detection, such as molecular beacons and TaqMan® 5′-3′ exonuclease chemistry (see for example U.S. Pat. Nos. 5,119,801, 5,312,728, 5,691,146; 5,723,591; 5,691,146; 5,876,930; and associated instrumentation). Other methods are available to detect the presence of hybridised oiigonucleotides to specific DNA sequences.
Many of these presently available methods require the use of expensive or potentially hazardous reagents, such as radioactive materials and fluorescently labeled oligonucleotides. In addition, many of the methods are time consuming and involve many steps that may ultimately reduce the accuracy and/or sensitivity of the method applied. As a consequence, the majority of these methods are poorly suited to the field of livestock SNP mass screening.
Other methods available utilise the proofreading capacity of DNA polymerases. Methods of determining the presence or absence of a predetermined nucleic acid target sequence in a nucleic acid sample using this proofreading activity are in part described in International Publication WO 00/49179 and International Publication WO 00/49182. One method described in these publications utilises a depolymerase enzyme that depolymerises nucleic acid from the 3′-terminus of an oligonucleotide probe hybridized to a nucleic acid target sequence via the process of pyrophosphorolysis and measures the subsequent release of one or more nucleotide triphosphates. In a second method described, the exonuclease activity of DNA polymerase is used to remove mismatched nucleotides from the 3′ end of a probe. To minimise the background present in the detection step of the method the hybridised probe/sample complex is purified from the remaining oligonucleotide.
The 3′-5′ exonuclease activity of DNA polymerases has been intensively studied since the late 1980's and it is known that this activity increases the accuracy of DNA replication over DNA polymerisation alone (Morales J C and Kool E T (2000) Biochemistry 39(10): 2626-32; Lam W C, Van der Schans E J, Joyce C M, Millar D P (1998) Biochemistry 37(6): 1513-22; Das S K and Fujimura R K (1980) Nucleic Acids Research 8(3): 657-71; Baker R P and Reha-Krantz L J (1998) Proc. Natl. Acad. Sci U.S.A 95(7): 3507-12; Canard b, Cardona B, Sarfati R S (1995) Proc. Natl. Acad. Sci U.S.A 94(24): 10859-63; de Vega M, Lazaro J M, Sala M, Blanco I (1996) EMBO J 15(5): 1182-92; Goodman M F, Creighton S, Bloom L B, Petruska J (1993) Crit Rev Biochem Mol Biol 28(2): 83-126; Johnson K A (1993) Annu Rev Biochem 62: 685-713; Thomas K R and Baldomero M O (1978) The Journal of Biological Chemistry 253(2): 424-429; Perler F B, Kumar S, Kong H (1996) Advances in Protein Chemistry 48: 377435.
However, the steps involved in proofreading by the 5′-3′ exonuclease activity are not well defined. Marquez L A and Reha-Krantz L J (1996) J Biol Chem. 271(46): 28903-11, studied the biochemical characteristics of a mutant T4 DNA polymerase to determine its role in strand separation. Using a mutant T4 DNA polymerase, together with the subsequent release of fluorescent d2APMP as a detection system, the molecular details of the 3′-5′ exonuclease activity of the T4 DNA polymerase were determined.
Using this method Marquez & Reha-Krantz were able to show how DNA with a single terminal mismatch was converted into a substrate for the hydrolysis reaction. From this study it was possible to determine the rate limiting steps required to prepare the DNA for the excision reaction. The researchers' findings showed the proofreading pathway in which DNA is first transferred from the polymerase active center to a pre-exonuclease complex and then further processed to produce the partially single-stranded DNA required for the hydrolysis reaction.
The present invention seeks to provide a low cost, high throughput detection system for identifying and characterizing individual animals on the basis of differences in individual nucleotides at particular positions in the genome (i.e. single nucleotide polymorphisms (SNPs)). It differs from and improves upon the previous methods which utilize the 3′-5′ exonuclease activity of DNA polymerases and other suitable exonucleases. Without exception, the prior methods rely on the use of DNA probes which can be also be used as DNA primers in PCR (referred to in International Publication WO 00/49179 and International Publication WO 00/49182 as primers). The method of the present invention requires rather that the oligonucleotide probes hybridised to the sample are not able to be extended by the action of a DNA polymerase during the cleavage stage of the reaction and cannot therefore be considered as primers for PCR. To this end the oligonucleotides are blocked at the 3′ end by the use of phosphate, dideoxy or other moieties that prevent extension of the oligonucleotide probe by the polymerase in the reaction. This distinction has the benefit that the method reaction can be carried out without the need for prior purification of the oligonucleotide probe/sample duplex as the polymerization reaction is prevented unless cleavage of the blocking 3′ nucleotide has occurred.
The main purpose of utilising this protocol and technology is its ability to lower the cost of individual ID tests, a very necessary feature for applications within livestock industries where mass screenings are generally the aim.
The preceding discussion of the background art is intended to facilitate an understanding of the present invention only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was part of the common general knowledge in Australia as at the priority date of the application.
Definitions
To facilitate understanding of the present invention, a number of terms are defined below.
A “probe” as used herein refers to an oligonucleotide or polynucleotide sequence which is able to hybridise to a sample DNA sequence under low stringency conditions, and which is blocked at the 3′ end such that it cannot be extended by DNA polymerases but is able to act as a substrate for the 3′-5′ proofreading exonuclease activity of DNA polymerases.
A “primer” as used herein refers to an oligonucleotide or polynucleotide sequence which is able to hybridise to a sample DNA sequence under low stringency conditions, and which can be extended by DNA polymerases with the concomitant release of pyrophosphate.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.